Gene delivery is a promising method for the treatment of acquired and inherited diseases. A number of viral-based systems for gene transfer purposes have been described, such as retroviral systems, which are currently the most widely used viral vector systems for this purpose. For descriptions of various retroviral systems, see, e.g., U.S. Pat. No. 5,519,740; Miller & Rosman, BioTechniques 7:980–990 (1989); Miller, Human Gene Therapy 1:5–14 (1990);Scarpa et al., Virology 180:849–852 (1991); Burns et al., Proc. Natl. Acad. Sci. USA 90:8033–8037 (1993); Boris-Lawrie & Temin, Cur. Opin. Genet. Develop. 3:102–109 (1993).
Adeno-associated virus (AAV) systems have also been used for gene delivery. AAV is generally considered a good choice for gene delivery because it has not been associated with any human or animal disease and does not appear to alter the biological properties of the host cell upon integration. AAV, which belongs to the genus Dependovirus, is a helper-dependent DNA parvovirus. Thus, in order for effective AAV virion production to occur, the host cell must also be infected with an unrelated helper virus, either adenovirus (Ad), a herpesvirus (HSV), or vaccinia virus. The helper virus supplies accessory functions that are necessary for most steps in AAV replication. In the absence of such infection, AAV establishes a latent state by insertion of its genome into a host cell chromosome. Subsequent infection by a helper virus rescues the integrated copy which can then replicate to produce infectious viral progeny. AAV has a wide host range and is able to replicate in cells from any species so long as there is also a successful infection of such cells with a suitable helper virus. For example, human AAV will replicate in canine cells co-infected with a canine adenovirus. For a review of AAV, see, e.g., Berns & Bohenzky, Advances in Virus Research 32:243–307 (Academic Press, Inc. 1987).
The AAV genome is composed of a linear single-stranded DNA molecule which contains 4681 bases (B ems & Bohenzky, supra). The genome includes inverted terminal repeats (ITRs) at each end which function in cis as origins of DNA replication and as packaging signals for the virus. The ITRs are approximately 145 bp in length. The internal nonrepeated portion of the genome includes two large open reading frames, known as the AAV rep and cap regions, respectively. These regions code for the viral proteins involved in replication and packaging of the virion. In particular, a family of at least four viral proteins are synthesized from the AAV rep region, Rep 78, Rep 68, Rep 52 and Rep 40, named according to their apparent molecular weight. The AAV cap region encodes at least three proteins, VP1, VP2 and VP3. For a detailed description of the AAV genome, see, e.g., Muzyczka, Current Topics in Microbiol. and Immunol. 158:97–129 (1992). For descriptions of the construction of recombinant AAV virions see, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publication Numbers WO 92/01070 (published 23 Jan. 1992) and WO 93/03769 (published 4 Mar. 1993); Lebkowski et al., Molec. Cell. Biol. 8:3988–3996 (1988); Vincent et al., Vaccines 90 (Cold Spring Harbor Laboratory Press 1990); Carter, Current Opinion in Biotechnology 3:533–539 (1992); Muzyczka, Current Topics in Microbiol. and Immunol. 158:97–129 (1992); Kotin, Human Gene Therapy 5:793–801 (1994).
Contemporary recombinant AAV (rAAV) virion production involves co-transfection of a host cell with an AAV vector plasmid usually containing one or more transgenes flanked by AAV ITRs, and a construct which provides AAV helper functions (e.g., rep and cap) to complement functions missing from the AAV vector plasmid. In this manner, the host cell is capable of expressing the AAV proteins necessary for AAV replication and packaging. To provide accessory functions, the host cell is then infected with a helper virus, typically an infectious adenovirus (type 2 or 5), or herpesvirus.
More particularly, AAV vector plasmids can be engineered to contain a functionally relevant nucleotide sequence of interest (e.g., a selected gene, antisense nucleic acid molecule, ribozyme, or the like) that is flanked by AAV ITRs which provide for AAV replication and packaging functions. After an AAV helper plasmid and an AAV vector plasmid bearing the nucleotide sequence are introduced into the host cell by transient transfection, the transfected cells can be infected with a helper virus, most typically an adenovirus, which, among other functions, transactivates the AAV promoters present on the helper plasmid that direct the transcription and translation of AAV rep and cap regions. Upon subsequent culture of the host cells, rAAV virions (harboring the nucleotide sequence of interest) and helper virus particles are produced.
When the host cell is harvested and a crude extract is produced, the resulting preparation will contain, among other components, approximately equal numbers of rAAV virion particles and infectious helper virions. rAAV virion particles can be purified away from most of the contaminating helper virus, unassembled viral proteins (from the helper virus and AAV capsid) and host cell proteins using known techniques.
Purified rAAV virion preparations that have been produced using infection with adenovirus type-2 contain high levels of contaminants. Particularly, 50% or greater of the total protein obtained in such rAAV virion preparations is free adenovirus fiber protein. Varying amounts of several unidentified adenoviral and host cell proteins are also present. Additionally, significant levels of infectious adenovirus virions are obtained, necessitating heat inactivation. The contaminating infectious adenovirus can be inactivated by heat treatment (56° C. for 1 hour) and rendered undetectable by sensitive adenovirus growth assays (e.g., by cytopathic effect (CPE) in a permissive cell line). However, heat treatment also results in an approximately 50% drop in the titer of functional rAAV virions.
Production of rAAV virions using an infectious helper virus (such as an adenovirus type-2, or a herpesvirus) to supply accessory functions is undesirable for several reasons. AAV vector production methods which employ a helper virus require the use and manipulation of large amounts of high titer infectious helper virus which presents a number of health and safety concerns, particularly in regard to the use of a herpesvirus. Selected herpes simplex virus type-1 (HSV-1) genes are significantly less efficient at supporting AAV replication than adenovirus- derived functions. Weindler et al., J. Virol. 65:2476–2483 (1991). In addition, some adenoviral proteins are cytotoxic or cytostatic to the host cell. For example, the E4ORF6 protein is toxic to cells in the presence of the E1B55k protein. Furthermore, concomitant production of helper virus particles in rAAV virion producing cells diverts large amounts of cellular resources away from rAAV virion production, possibly resulting in lower rAAV virion yields.
More particularly, in methods where infection of cells with adenovirus type-2 are used to provide the accessory functions, more than 95% of the contaminants found in the purified rAAV virion preparations are derived from adenovirus. The major contaminant, free adenovirus fiber protein, tends to co-purify with rAAV virions on CsCl density gradients due to a non-covalent association between the protein and rAAV virions. This association makes separation of the two especially difficult, lowering rAAV virion purification efficiency. Such contaminants may be particularly problematic since many adenoviral proteins, including the fiber protein, have been shown to be cytotoxic (usually at high concentrations), and thus may adversely affect or kill target cells. Thus, a method of producing rAAV virions without the use of infectious helper viruses to provide necessary accessory functions would be advantageous.
Because of the problems associated with the use of complete helper viruses, a number of researchers have investigated the genetic basis of accessory functions, particularly adenovirus- derived functions, in an attempt to derive functional helper constructs. Although many of the adenovirus (“Ad”) or herpes simplex virus (“HSV”) genes are incompletely mapped, it is known that Ad “early” genes are expressed before both the genes encoding the proteins necessary for replication and before the “late” genes. The early genes are divided into the following groups:
E1, E2, E4 and the VA RNAs. E1 is approximately 6 kb in size and encodes the E1A protein, the E1B19k protein, the E1B55k protein, and protein IX. A 72 kd E2A protein is encoded within E2, while E4ORF6 is encoded within the E4 region. It has been established that the E1B55k protein binds to both E4ORF6 and p53. Furthermore, the E4ORF6 protein is cytotoxic, but only in the presence of E1B55k.
It has been demonstrated that the full-complement of adenovirus genes are not required for accessory helper functions. In particular, adenovirus mutants incapable of DNA replication and late gene synthesis have been shown to be permissive for AAV replication. Ito et al., J. Gen. Virol. 9:243 (1970); Ishibashi et al, Virology 45:317 (1971). Similarly, mutants within the E2B and E3 regions have been shown to support AAV replication, indicating that the E2B and E3 regions are probably not involved in providing accessory functions. Carter et al., Virology 126:505 (1983). However, adenoviruses defective in the E1 region, or having a deleted E4 region, are unable to support AAV replication. Thus, E1A and E4 regions are likely required for AAV replication, either directly or indirectly. Laughlin et al., J. Virol. 41:868 (1982); Janik et al., Proc. Natl. Acad. Sci. USA 78:1925 (1981); Carter et al. (1983), supra). Other characterized Ad mutants include: E1B (Laughlin et al. (1982), supra; Janik et al. (1981), supra; Ostrove et al., Virology 104:502 (1980)); E2A (Handa et al., J. Gen. Virol. 29:239 (1975); Strauss et al., J. Virol. 17:140 (1976); Myers et al., J. Virol. 35:665 (1980); Jay et al., Proc. Natl. Acad. Sci. USA 78:2927 (1981); Myers et al., J. Biol. Chem. 256:567 (1981)); E2B (Carter, Adeno-Associated Virus Helper Functions, in I CRC Handbook of Parvoviruses (P. Tijssen ed., 1990)); E3 (Carter et al. (1983), supra); and E4 (Carter et al.(1983), supra; Carter (1995)). Although studies of the accessory functions provided by adenoviruses having mutations in the E1B coding region have produced conflicting results, Samulski et al., J. Virol. 62:206–210 (1988), recently reported that E1B55k is required for AAV virion production, while E1B19k is not. In addition, International Publication WO 97/17458 and Matshushita et al., Gene Therapy 5:938–945 (1998), describe accessory function vectors encoding various Ad genes.
Further characterization of the Ad genes required for helper functions has been attempted by transfecting various regions of the Ad genome and assessing virion production. Particularly, in vitro AAV replication has been assessed using human 293 cells transiently transfected with various combinations of adenovirus restriction fragments encoding single adenovirus genes or groups of genes. Janik et al. (1981), supra. Initial transfection studies were done in cells that stably express the adenovirus E1A and E1B regions, so the requirement for those regions could not be tested. However, it was deduced that the combination of three adenoviral gene regions, VA RNA, E2A and E4, could provide accessory functions (e.g., support AAV replication) at a level that was substantially above background, but that was still approximately 8,000 fold below the level provided by infection with adenovirus. When all combinations of two of the three genes were tested, the accessory function levels ranged between 10,000 to 100,000 fold below the levels provided by infection with adenovirus.
Accordingly, there remains a need in the art to identify a subset of the adenovirus genome or functional homologues of the adenovirus genome, that include only those accessory functions required for AAV vector production. Furthermore, if the required subset includes cytotoxic genes, there remains a need to control expression of these genes and resulting levels of the toxic gene product. The identification of the minimal complement of genes and modifications to control expression can be used to design constructs which, when introduced into a suitable cell line, allow for the selection of an AAV packaging cell line.